Live cell imaging by multifocal multiphoton microscopy

Multifocal multiphoton microscopy (MMM) permits parallel multiphoton excitation by scanning an array of high numerical aperture foci across a plane in the sample. MMM is particularly suitable for live cell investigations since it combines advantages of standard multiphoton microscopy such as optical sectioning and suppression of out-of-focus phototoxicity with high recording speeds. Here we describe several applications of MMM to live cell imaging using the neuroendocrine cell line PC12 and bovine chromaffin cells. Stainings were performed with the acidophilic dye acridine orange and the lipophilic dyes FM1-43 and Fast DiA as well as by transfection of the cells with GFP. In both bovine chromaffin and PC12 cells structural elements of nuclear chromatin and the 3-D distribution of acidic organelles inside the cells were visualized. In PC12 cells differentiated by nerve growth factor examples of neurites were monitored. Stainings of membranes were used to reconstruct the morphology of cells and neurites in three dimensions by volume-rendering and by isosurface plots. 3-D reconstructions were composed from stacks of about 50 images each with a diameter of 30-100 microm that were acquired within a few seconds. We conclude that MMM proves to be a technically simple and very effective method for fast 3-D live cell imaging at high resolution.     

The first decade of using multiphoton microscopy for high-power kidney imaging

In this review, we highlight the major scientific breakthroughs in kidney research achieved using multiphoton microscopy (MPM) and summarize the milestones in the technological development of kidney MPM during the past 10 years. Since more and more renal laboratories invest in MPM worldwide, we discuss future directions and provide practical, useful tips and examples for the application of this still-emerging optical sectioning technology. Advantages of using MPM in various kidney preparations that range from freshly dissected individual glomeruli or the whole kidney in vitro to MPM of the intact mouse and rat kidney in vivo are reviewed. Potential combinations of MPM with micromanipulation techniques including microperfusion and micropuncture are also included. However, we emphasize the most advanced and complex, quantitative in vivo imaging applications as the ultimate use of MPM since the true mandate of this technology is to look inside intact organs in live animals and humans.     

Simultaneous multiple-excitation multiphoton microscopy yields increased imaging sensitivity and specificity

Background  

Multiphoton microscopy (MPM) offers many advantages over conventional wide-field and confocal laser scanning microscopy (CLSM) for imaging biological samples such as 3D resolution of excitation, reduced phototoxicity, and deeper tissue imaging. However, adapting MPM for critical multi-color measurements presents a challenge because of the largely overlapping two-photon absorption (TPA) peaks of common biological fluorophores. Currently, most multi-color MPM relies on the absorbance at one intermediate wavelength of multiple dyes, which introduces problems such as decreased and unequal excitation efficiency across the set of dyes.     

New developments in multiphoton microscopy

Multiphoton laser-scanning microscopy is still developing rapidly, both technologically and by broadening its range of application. Technical progress has been made in the optimization of fluorophores, in increasing the imaging depth of multiphoton microscopy, and in microscope miniaturization. These advances further facilitate the study of neuronal structure and signaling in living and even in behaving animals, in particular in combination with the expression of fluorescent proteins. In addition, nonlinear optical contrast mechanisms other than multiphoton excitation of fluorescence are being explored.     

Volumetric imaging of erythrocytes using label‐free multiphoton photoacoustic microscopy

Photoacoustic microscopy (PAM) is an imaging modality well suited to mapping vasculature and other strong absorbers in tissue. However, one of the primary drawbacks to PAM when used for high‐resolution imaging is the relatively poor axial resolution due to the inverse dependence on the transducer bandwidth. While submicron lateral resolution PAM can be achieved by tightly focusing the excitation light, the axial resolution is fundamentally limited to 10s of microns for typical transducer frequencies. Here we present a multiphoton PAM technique called transient absorption ultrasonic microscopy (TAUM), which results in a completely optically resolved voxel with an experimentally measured axial resolution of 1.5 microns. This technique is demonstrated by imaging individual red blood cells in three dimensions in blood smear and ex vivo tissues. To the best of our knowledge, this is the first demonstration of fully resolved, volumetric photoacoustic imaging of erythrocytes. (© 2014 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Maintaining polarization in polarimetric multiphoton microscopy

Polarimetric measurements in multiphoton microscopy can reveal information about the local molecular order of a sample. However, the presence of a dichroic through which the excitation beam propagates will generally scramble its polarization. We propose a simple scheme whereby a second properly‐oriented compensation dichroic is used to negate any alteration regardless of the wavelength and the initial polarization. We demonstrate how this robust and rapid approach simplifies polarimetric measurements in second‐harmonic generation, two‐photon excited fluorescence and coherent anti‐Stokes Raman scattering.

Illustration of the polarization maintaining strategy with the compensating dichroic oriented such that its s‐ and p‐axes are interchanged with these of the primary dichroic.     

Temporal focusing‐based widefield multiphoton microscopy with spatially modulated illumination for biotissue imaging

A developed temporal focusing‐based multiphoton excitation microscope (TFMPEM) has a digital micromirror device (DMD) which is adopted not only as a blazed grating for light spatial dispersion but also for patterned illumination simultaneously. Herein, the TFMPEM has been extended to implement spatially modulated illumination at structured frequency and orientation to increase the beam coverage at the back‐focal aperture of the objective lens. The axial excitation confinement (AEC) of TFMPEM can be condensed from 3.0 μm to 1.5 μm for a 50 % improvement. By using the TFMPEM with HiLo technique as two structured illuminations at the same spatial frequency but different orientation, reconstructed biotissue images according to the condensed AEC structured illumination are shown obviously superior in contrast and better scattering suppression. Picture : TPEF images of the eosin‐stained mouse cerebellar cortex by conventional TFMPEM (left), and the TFMPEM with HiLo technique as 1.09 μm^?1 spatially modulated illumination at 90° (center) and 0° (right) orientations.

     

In vivo brain imaging of mitochondrial Ca2+ in neurodegenerative diseases with multiphoton microscopy

Mitochondria are involved in a large number of essential roles related to neuronal function. Ca²⁺ handling by mitochondria is critical for many of these functions, including energy production and cellular fate. Conversely, mitochondrial Ca²⁺ mishandling has been related to a variety of neurodegenerative diseases. Investigating mitochondrial Ca²⁺ dynamics is essential for advancing our understanding of the role of intracellular mitochondrial Ca²⁺ signals in physiology and pathology. Improved Ca²⁺ indicators, and the ability to target them to different cells and compartments, have emerged as useful tools for analysis of Ca²⁺ signals in living organisms. Combined with state-of-the-art techniques such as multiphoton microscopy, they allow for the study of mitochondrial Ca²⁺ dynamics in vivo in mouse models of the disease. Here, we provide an overview of the Ca²⁺ transporters/ion channels in mitochondrial membranes, and the involvement of mitochondrial Ca²⁺ in neurodegenerative diseases followed by a summary of the main tools available to evaluate mitochondrial Ca²⁺ dynamics in vivo using the aforementioned technique.     

In vivo multiphoton microscopy of melasma

Melasma is a skin disorder characterized by hyperpigmented patches due to increased melanin production and deposition. In this pilot study, we evaluate the potential of multiphoton microscopy (MPM) to characterize non‐invasively the melanin content, location, and distribution in melasma and assess the elastosis severity. We employed a clinical MPM tomograph to image in vivo morphological features in melasma lesions and adjacent normal skin in 12 patients. We imaged dermal melanophages in most dermal melasma lesions and occasionally in epidermal melasma. The melanin volume fraction values measured in epidermal melasma (14% ± 4%) were significantly higher (p < 0.05) than the values measured in perilesional skin (11% ± 3%). The basal keratinocytes of melasma and perilesions showed different melanin distribution. Elastosis was predominantly more severe in lesions than in perilesions and was associated with changes in melanin distribution of the basal keratinocytes. These results demonstrate that MPM may be a non‐invasive imaging tool for characterizing melasma.     

When multiphoton microscopy sees near infrared

The need for quantification and real time visualization of developmental processes has called for increasingly sophisticated imaging techniques. Among them, multiphoton microscopy reveals itself to be an extremely versatile tool owing to its unique ability to combine fluorescent imaging, laser ablation, and higher harmonic generation. Furthermore, recent advances in femtosecond lasers and optical parametric oscillators (OPO) are now opening doors for imaging at unprecedented wavelengths centered in the tissue transparency window. This Review describes promising multiphoton approaches using OPO and the growing number of useful applications of non-linear microscopy in the field of developmental biology. Basic characteristics associated with these techniques are described along with the main experimental challenges when applied to embryo imaging.     

Immunoelectron microscopy in embryos

Immunogold labeling of proteins in sections of embryos embedded in acrylate media provides an important analytical tool when the resolving power of the electron microscope is required to define sites of protein function. The protocol presented here was established to analyze the role and dynamics of the activated protein kinase C/Rack1 regulatory system in the patterning and outgrowth of limb bud mesenchyme. With minor changes, especially in the composition of the fixative solution, the protocol should be easily adaptable for the postembedding immunogold labeling of any other antigen in tissues of embryos of diverse species. Quantification of the labeling can be achieved by using electron microscope systems capable of supporting digital image analysis.     

Nonlinear magic: multiphoton microscopy in the biosciences

Multiphoton microscopy (MPM) has found a niche in the world of biological imaging as the best noninvasive means of fluorescence microscopy in tissue explants and living animals. Coupled with transgenic mouse models of disease and 'smart' genetically encoded fluorescent indicators, its use is now increasing exponentially. Properly applied, it is capable of measuring calcium transients 500 microm deep in a mouse brain, or quantifying blood flow by imaging shadows of blood cells as they race through capillaries. With the multitude of possibilities afforded by variations of nonlinear optics and localized photochemistry, it is possible to image collagen fibrils directly within tissue through nonlinear scattering, or release caged compounds in sub-femtoliter volumes.     

Functional studies in living animals using multiphoton microscopy

In vivo microscopy is a powerful method for studying fundamental issues of physiology and pathophysiology. The recent development of multiphoton fluorescence microscopy has extended the reach of in vivo microscopy, supporting high-resolution imaging deep into the tissues and organs of living animals. As compared with other in vivo imaging techniques, multiphoton microscopy is uniquely capable of providing a window into cellular and subcellular processes in the context of the intact, functioning animal. In addition, the ability to collect multiple colors of fluorescence from the same sample makes in vivo microscopy uniquely capable of characterizing up to three parameters from the same volume, supporting powerful correlative analyses. Since its invention in 1990, multiphoton microscopy has been increasingly applied to numerous areas of medical investigation, providing invaluable insights into cell physiology and pathology. However, researchers have only begun to realize the true potential of this powerful technology as it has proliferated beyond the laboratories of a relatively few pioneers. In this article we present an overview of the advantages and limitations of multiphoton microscopy as applied to in vivo imaging. We also review specific examples of the application of in vivo multiphoton microscopy to studies of physiology and pathology in a variety of organs including the brain, skin, skeletal muscle, tumors, immune cells, and visceral organs.     

Picosecond multiphoton scanning near-field optical microscopy.

We have implemented simultaneous picosecond pulsed two- and three-photon excitation of near-UV and visible absorbing fluorophores in a scanning near-field optical microscope (SNOM). The 1064-nm emission from a pulsed Nd:YVO4 laser was used to excite the visible mitochondrial specific dye MitoTracker Orange CM-H2TMRos or a Cy3-labeled antibody by two-photon excitation, and the UV absorbing DNA dyes DAPI and the bisbenzimidazole BBI-342 by three-photon excitation, in a shared aperture SNOM using uncoated fiber tips. Both organelles in human breast adenocarcinoma cells (MCF 7) and specific protein bands on polytene chromosomes of Drosophila melanogaster doubly labeled with a UV and visible dye were readily imaged without photodamage to the specimens. The fluorescence intensities showed the expected nonlinear dependence on the excitation power over the range of 5-40 mW. An analysis of the dependence of fluorescence intensity on the tip-sample displacement normal to the sample surface revealed a higher-order function for the two-photon excitation compared to the one-photon mode. In addition, the sample photobleaching patterns corresponding to one- and two-photon modes revealed a greater lateral confinement of the excitation in the two-photon case. Thus, as in optical microscopy, two-photon excitation in SNOM is confined to a smaller volume.     

Automatic imaging of Drosophila embryos with light sheet fluorescence microscopy on chip

We present a microscope on chip for automated imaging of Drosophila embryos by light sheet fluorescence microscopy. This integrated device, constituted by both optical and microfluidic components, allows the automatic acquisition of a 3D stack of images for specimens diluted in a liquid suspension. The device has been fully optimized to address the challenges related to the specimens under investigation. Indeed, the thickness and the high ellipticity of Drosophila embryos can degrade the image quality. In this regard, optical and fluidic optimization has been carried out to implement dual-sided illumination and automatic sample orientation. In addition, we highlight the dual color investigation capabilities of this device, by processing two sample populations encoding different fluorescent proteins. This work was made possible by the versatility of the used fabrication technique, femtosecond laser micromachining, which allows straightforward fabrication of both optical and fluidic components in glass substrates.     

Quantitative high-speed imaging of entire developing embryos with simultaneous multiview light-sheet microscopy

Live imaging of large biological specimens is fundamentally limited by the short optical penetration depth of light microscopes. To maximize physical coverage, we developed the SiMView technology framework for high-speed in vivo imaging, which records multiple views of the specimen simultaneously. SiMView consists of a light-sheet microscope with four synchronized optical arms, real-time electronics for long-term sCMOS-based image acquisition at 175 million voxels per second, and computational modules for high-throughput image registration, segmentation, tracking and real-time management of the terabytes of multiview data recorded per specimen. We developed one-photon and multiphoton SiMView implementations and recorded cellular dynamics in entire Drosophila melanogaster embryos with 30-s temporal resolution throughout development. We furthermore performed high-resolution long-term imaging of the developing nervous system and followed neuroblast cell lineages in vivo. SiMView data sets provide quantitative morphological information even for fast global processes and enable accurate automated cell tracking in the entire early embryo.     

Insight into plant cell wall chemistry and structure by combination of multiphoton microscopy with Raman imaging

Spontaneous Raman scattering microspectroscopy, second harmonic generation (SHG) and 2‐photon excited fluorescence (2PF) were used in combination to characterize the morphology together with the chemical composition of the cell wall in native plant tissues. As the data obtained with unstained sections of Sorghum bicolor root and leaf tissues illustrate, nonresonant as well as pre‐resonant Raman microscopy in combination with hyperspectral analysis reveals details about the distribution and composition of the major cell wall constituents. Multivariate analysis of the Raman data allows separation of different tissue regions, specifically the endodermis, xylem and lumen. The orientation of cellulose microfibrils is obtained from polarization‐resolved SHG signals. Furthermore, 2‐photon autofluorescence images can be used to image lignification. The combined compositional, morphological and orientational information in the proposed coupling of SHG, Raman imaging and 2PF presents an extension of existing vibrational microspectroscopic imaging and multiphoton microscopic approaches not only for plant tissues.      

Label-free imaging of lipid-droplet intracellular motion in early Drosophila embryos using femtosecond-stimulated Raman loss microscopy

Lipid droplets are complex organelles that exhibit highly dynamic behavior in early Drosophila embryo development. Imaging lipid droplet motion provides a robust platform for the investigation of shuttling by kinesin and dynein motors, but methods for imaging are either destructive or deficient in resolution and penetration to study large populations of droplets in an individual embryo. Here we report real-time imaging and quantification of droplet motion in live embryos using a recently developed technique termed "femtosecond-stimulated Raman loss" microscopy. We captured long-duration time-lapse images of the developing embryo, tracked single droplet motion within large populations of droplets, and measured the velocity and turning frequency of each particle at different apical-to-basal depths and stages of development. To determine whether the quantities for speed and turning rate measured for individual droplets are sufficient to predict the population distributions of droplet density, we simulated droplet motion using a velocity-jump model. This model yielded droplet density distributions that agreed well with experimental observations without any model optimization or unknown parameter estimation, demonstrating the sufficiency of a velocity-jump process for droplet trafficking dynamics in blastoderm embryos.